The field of tissue engineering relates to the technology of generating molecules, cells, tissues and, in rare instances, complete organs suitable for regenerating phenotypically specific tissue in host defects or injuries. Fundamental to the success of these efforts is understanding the mechanisms of cellular tensegrity by which undifferentiated pluripotent mesenchymal cells interpret information they receive from their microenvironments and translate these signals into biochemical messages capable of influencing expression of the genome.
Traditional in vitro cell culture methods employ two dimensional, polystyrene cell culture plates. While much valuable information has been discovered in such systems, two significant issues limit their application to tissue engineering problems. First, in vitro culture plates are generally rigid structures made, typically, of polystyrene. Secondly, in vitro culture plates provide the cells with only a two dimensional substratum. This two dimensional substratum is restrictive, since cells of tissues and organs respond to signals initiated within a three dimensional (3-D) microenvironment. Cells comprising all tissues, including bone, have some capacity to alter their three dimensional morphologies in response to changes in mechanical forces present within their environments.
Tissue engineering research has focused on the regeneration of bone and articular cartilage. Some remarkable successes toward these ends have been achieved by employing bioresorbable, synthetic compounds to fabricate anatomically and/or functionally specific, 3-D architectures by which biologically active agents and/or living cells are presented to bone or cartilage defects. One approach involves isolating cells from the body, expanding them in in vitro cultures, placing them on or within structural matrices, and implanting the new system inside the body. A commercial example of this approach is Carticell® (Genzyme Corp., Boston, Mass.), wherein a process and device that expands a patient's own articular cartilage cells (chondrocytes) in vitro and return them within a type I collagen matrix for treatment of articular cartilage defects. Another commercial construct known as Infuse® (Medtronic-Softmore-Danek, Memphis, Tenn.) is a device composed of a type I collagen sponge saturated with a protein known as bone morphogenetic protein (recombinant human bone morphogenetic protein-2 [rh-BMP-2]). This saturated sponge induces bone formation within the interbody spinal fusion model in the human.
Tissue engineering research has been directed toward: identification of appropriate cell sources (mature, pluripotent progenitor and stem cells); selection of suitable bioactive molecules (growth factors and morphogens); and fabricating devices of various compositions and geometries (synthetic or natural polymers, meshes or foams) to function as cell culture substrata. A three dimensional substratum may be described as an intricate biocompatible and bioresorbable network of natural or synthetic fibers defining an internal organization of spaces (voids) within which cells can grow, migrate, proliferate and differentiate if they are provided with a suitable extracellular matrix (ECM), and appropriately formulated nutrient media either in vitro or in vivo.
Adult bone marrow stromal cells, and their mesenchymal stem cell (MSC) progeny, have been shown to be pluripotent stem cells, capable of differentiating into cells of liver (hepatocyte), bone (osteoblast), fat (adipocyte), cartilage (chondrocyte), myocardium (cardiomyocyte), and the neuron.
Mesenchymal stem cells (MSC) are the formative pluripotential blast cells found inter alia in association with capillaries (i.e., the vascular pericyte) and bone marrow that are capable of differentiating into any of the specific types of connective tissue cells such as adipocytes, osteoblasts, chondrocytes, fibroblasts and myocytes (of smooth and skeletal muscle as well as the cardiomyocyte). Phenotype selection for the MSC is directed by various influences exerted by soluble bioactive factors such growth factors, morphogens and cytokines as well as information derived from their microenvironments by means of mechanochemical signal transduction. Although these cells are normally present at very low frequencies in bone marrow, through a process disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 these cells may be isolated, purified and replicated in culture.
In order to isolate human mesenchymal stem cells (h-MSC), it is necessary to isolate rare pluripotent mesenchymal stem cells from other cells in the bone marrow or other MSC sources. Bone marrow cells may be obtained from the iliac crest, femur, tibia, spine, rib or other medullary spaces. Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood and other mesenchymal stem cell tissues.
Isolated human mesenchymal stem cell (h-MSC) compositions serve as progenitors for various mesenchymal cell lineages. Isolated mesenchymal stem cell populations have the ability to expand in culture without differentiating and have the ability to differentiate along specific connective tissue lineages when cultured in vitro or introduced in vivo at a place of damaged tissue. In order to realize the therapeutic potential of these cells to restore diseased or damaged connective tissues, they must be entrusted to a delivery vehicle possessed of biochemical and mechanical properties appropriate for propagation of a particular cell phenotype.
Several patents have attempted to describe a suitable biocompatible delivery vehicle for both undifferentiated and phenotypically mature cells. For example, U.S. Pat. No. 6,482,231 discloses a biological material for the repair of connective tissues comprising: a) a cell preparation enriched with mesenchymal stem cells, b) three-dimensional extracellular matrix comprising a hyaluronic acid derivative. This reference discloses the various ways to chemically treat hyaluronic acid to alter its biophysical and biological properties. For instance, U.S. Pat. No. 6,482,231 discloses treatment with formaldehyde or vinyl sulfone to give rise to cross-linked gels. In addition, various hyaluronic acid derivatives are disclosed, for example a partial or complete ester of hyaluronic acid with an aliphatic, aromatic or araliphatic alcohol, and a crosslinked hyaluronic acid derivative.
U.S. Pat. No. 6,596,274 discloses a biological material comprising two components, wherein the first component comprises alternatively (1) a culture of autologous or homologous bone marrow stem cells partially or completely differentiated into specific connective tissue cellular lines or (2) a sole extracellular matrix free from any cellular component secreted by the specific connective tissue cellular lines; and a second component comprises a three-dimensional biocompatible and biodegradable matrix consisting of a hyaluronic acid ester having a degree of esterification between 25 and 100%. The preferred hyaluronic acid ester disclosed is the benzyl alcohol ester having a degree of esterification varying from 25 to 100%.
U.S. Pat. No. 5,166,187 discloses a biomaterial consisting of an association of collagen, chitosan acetylated to a degree of acetylation between about 10% and about 40% and of glycosaminoglycans. The disclosed biomaterial is used for making extracellular matrices for regeneration of nerve cells and bones as well as biocompatible envelopes. A particular application is the making of artificial skin consisting of a dermal layer.
European Patent EP 1003567 B1 discloses a polysaccharide based gel which comprises: (1) chitosan or a chitosan derivative; and (2) a salt of polyol or sugar. The gel may be formed in situ within a tissue, organ or cavities of an animal or human.
European Patent Application EP 0784985 A1 discloses a bioabsorbable hydrophilic material comprising one or more compounds selected from a group consisting of gelatin, collagen, a collagen derivative, chitosan, a chitosan derivative, and triethanolamine alginate. A bone-forming graft is also disclosed comprising a bone morphogenetic protein and the bioabsorbable hydrophilic material.
European Patent Application EP 0544259 A1 discloses a water insoluble biocompatible hyaluronic acid polyion complex that comprises hyaluronic acid and at least one biocompatible high molecular compound having amino or imino groups. The polyionic complex is made by reacting an alkalimetal salt of hyaluronic acid with the high molecular compound in an organic acid aqueous solution.
PCT patent WO 03/008007 A2 discloses an implantable device for facilitating the healing of voids in bone, cartilage and soft tissue. The device includes a cartilage region comprising a polyelectrolytic complex joined with a subchondral bone region. Each of these regions comprise a macrostructure of a bioresorbable polymer. The device also includes a microstructure which is composed of various polysaccharides including hyaluronic acid. The polyelectrolytic complex transforms to hydrogel, following the implant procedure.
The present disclosure describes the use of a malleable cell culture matrix comprising both hyaluronan and chitosan as well as a polyelectrolytic complex (PEC) of the two constituents, that are combined in their dry states prior to the formation of the polyelectrolyte complex. The disclosure describes a new material that can function in vitro as an malleable cell culture material for pluripotent cells and subsequently perform as the delivery vehicle for implantation of these same cells into a host tissue. The present application refers to “hyaluronan” and “hyaluronic acid” as synonyms and both terms will therefore be used interchangeably.